The pattern of cell divisions as it relates to the establishment the axes of embryonic symmetry i the sea urchin embryo is a major interest to our laboratory. While we have obtained useful information from intact, fixed embryos with confocal microscopic optical sectioning and computer graphic reconstructions, a more dynamic view is required. This necessitates obtaining data from living embryos, rather than the disjunct, static data obtained from fixed material. Our goal is to supravitally stain embryos for DNA, and obtain optical sections through the embryo over an extended time period. From these time-lapse data, three dimensional reconstructions can be prepared and a single cell and its progeny can be tracked through multiple nuclear division cycles and its division rate, orientation, size and shape related to other cells in the observed population. The only available fluorochromes that stain DNA are non-toxic to sea urchin embryos are the bisbenzimides,which are excitable by ultraviolet (UV) light. Unfortunately, UV irradiation damages the embryos. We propose to develop the technology of two-photon fluorophore excitation laser scanning microscopy as a solution to this problem, using the sea urchin embryo as a test system. With this technique, red (rather than UV) light is employed to excite a UV dye with the premise that light of longer wavelengths is less biologically damaging. Pilot experiments with sea urchin embryos have demonstrated the feasibility of this technology in the short term but more information is necessary. A series of experiments has been designed to further develop the optical and biological aspects of two-photon excitation and test its effects upon living biological specimens. If we are successful, this technology will provide an alternative means by which time-resolved, three-dimensional views of ongoing physiological processes can be obtained from living cells. Additionally, a variety of UV excitable fluorophores, that are currently not practicable for use with laser scanning confocal microscopy, will become available for use with living cells and tissues.